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==Shotgun library construction== Single-end sequencing libraries ===PCR with dUTP=== x 4 x 4 Template: 1ul 2x EconoTaq Master Mix 50ul 800 100uM AmpF6.3NH2 0.2ul 3.2 100uM AmpR6.3NH2 0.2ul 3.2 10mM dUTP 0.4ul 6.4 50x SYBG I 0.4ul 6.4 H2O 48ul 770 94C 3min -> 11 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C Purified the amplicon with one Qiaquick column each A: 82ng/ul x 30ul B: 51ng/ul x 30ul C: 59ng/ul x 30ul D: 85ng/ul x 30ul ===USER digestion=== Qiaquick purified DNA 15ul USER enzyme 2ul (3 ul used for tube a and d) 37C 1h ===S1 nuclease digestion=== 10 x S1 nuclease buffer: 2ul (2.3ul used for tube a and d) DNA after USER digestion: 17ul S1 nuclease (10U/ul): 2ul ddH2O 2ul 37C 15mins Purified with MinElute cloumn. Elute in 18ul H2O. a: 30ng/ul b: 20ng/ul c: 24ng/ul d: 23ng/ul ===End repair (epicentre)=== Fragmented DNA 15ul dNTP 2.5ul 10x buffer 2.5ul enzyme 0.5ul ATP 5ul Incubate at RT for 40 minutes Purified with minelute column and elute in 20ul Select the ~100bp fragments with TBE gel [[Image:ZhangLab_2 2009-05-24 17hr 57min.jpg]] ===Adaptor ligation=== DNA 20ul 2X Rapid Ligation buffer 27ul 100uM Solexa_1 adaptor 1ul 100uM Solexa_2 adaptor 1ul QuickLigase(400U/ul) 2ul Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns and elute in 16ul EB. performed one round of size selection on positive control tube using 2%SizeSelect E-gel. The total amount obtained is 25ul. ===Nick translation=== DNA 15ul 10x ThermoPol buffer 2ul (3ul for positive control) 10mM dNTP 0.4ul BSA 2ul Bst Pol (8U/ul) 1ul 65C 20min ===PCR=== x 5 DNA 10ul Solexa_PCR_up(100uM) 0.2ul Solexa_PCR_lo(100uM) 0.2ul 2x iProof master mix 50ul 50x SYBGI 0.4ul ddH2O 40ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec) 6 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold. [[Image:ZhangLab_2 2009-05-25 01hr 10min.jpg]] x4 DNA 10ul (second half of the products) Solexa_PCR_up(100uM) 0.2ul Solexa_PCR_lo(100uM) 0.2ul 2x iProof master mix 50ul 50x SYBGI 0.4ul ddH2O 40ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec) 5 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold. Purify with one qiaquick column for each set, and elute in 40ul EB a: 142.9ng/ul b: 101.2ng/ul c: 117.2ng/ul d: 126.5ng/ul perform gel size selection at 150-160bp Ethanol precipitation on the sets from above, and also four tubes obtained from Dr. Zhang. Elute in 40ul of ddH2O each. ===result=== gel quantification: 1: 2ul low mass ladder + 2ul H2O + 2ul loading dye 2: 1ul low mass ladder + 2ul H2O + 2ul loading dye 3-6: 1ul DNA + 3ul H2O + 2ul loading dye 7-10: 1ul DNA + 3ul H2O + 2ul loading dye [[Image:ZhangLab_2 2009-05-25 12hr 29min.jpg]] yield: a-k: 1.13 ng/ul = 10.71nM b-k: 1.45 ng/ul = 13.73nM c-k: 0.77 ng/ul = 7.27nM d-k: 5.2 ng/ul = 49.25nM a-A: 16.38 ng/ul = 155.1nM b-A: 14.09 ng/ul = 133.41nM c-A: 8.14 ng/ul = 77.1nM d-A: 12.25 ng/ul = 116.04nM A total of 8 tubes of solexa sequencing libraries were sent to BioGem. (calculated by Dr. Zhang) [tube 1]: PGP1_062509k, 19nM, 40ul [tube 2]: PGP2_062509k, 20nM, 40ul [tube 3]: PGP3_062509k, 19nM, 40ul [tube 4]: NA12878_062509k, 33nM, 40ul [tube 5]: PGP1_062509a, 77nM, 40ul [tube 6]: PGP2_062509a, 64nM, 40ul [tube 7]: PGP3_062509a, 49.5nM, 40ul [tube 8]: NA12878_062509a, 60nM, 40ul
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